Project Summary Non-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver function tests in the United States and is a major cause of cirrhosis, hepatocellular carcinoma and death. Although fatty liver disease is associated with the metabolic syndrome, the pathogenesis remains poorly understood. In the liver, the fidelity for protein folding is imperfect, and therefore excess misfolded or unfolded proteins may accumulate, a condition termed ER stress. The Unfolded Protein Response (UPR) is a protective signaling response present in all eukaryotic cells, and human and animal studies indicate that the UPR is important in NAFLD and its progressive sub-type of non-alcoholic steatohepatitis (NASH). However, the role and mechanisms of UPR signaling in the pathogenesis of NASH remains poorly understood. The UPR consists of three pathways that are activated by the ER proteins IRE1?, PERK and ATF6. These proteins serve both as sensors of ER stress and activators of their signaling pathways. In the canonical adaptive IRE1? pathway, activated p-IRE1? splices XBP1 into the active transcription factor XBP1s, which then activates protective downstream target genes. More recent data indicates that IRE1? has a major role in cell fate determination, with both the adaptive XBP1 pathway, and apoptotic Regulated Ire1?-Dependent Decay (RIDD) and TRAF2 pathways. In addition, IRE1? interacts with the UPR pathway involved with eIF2?/ATF4/CHOP signaling and other cellular stress signaling pathways. Although dysregulation of liver IRE1? and e-IF2? pathways have been associated with human NASH, the causative and mechanistic role of IRE1? in the pathogenesis of steatohepatitis remain poorly understood. The long-term goals of these studies are to enhance our understanding of the role of UPR signaling in the pathogenesis and progression of NASH. The objectives of this grant proposal are to further define the role of hepatic IRE1? adaptive and apoptotic signaling in the pathogenesis of non-alcoholic steatohepatitis. We have previously demonstrated that when fed a high fat diet, mice with hepatocyte-specific deletion of Xbp1 have increased susceptibility to develop NASH. We now propose three Specific Aims to further investigate the role of IRE1? signaling in the pathogenesis of steatohepatitis: to determine that enhanced hepatic IRE1? signaling increases the susceptibility to develop steatohepatitis using liver-specific XBP1(-/-) and liver-specific IRE1?(-/-) mice fed high fat diets (Aim 1); to delineate the regulation of apoptosis by hepatic IRE1? via the TRAF2, eIF2? and RIDD pathways (Aim 2); and to utilize liver-specific cell cultures and cultured hepatocytes to determine the role of IRE1? signaling in cell fate determination during hepatocyte lipotoxicity (Aim 3). By better understanding this IRE1?-mediated adaptive and apoptotic signaling in the liver, it may be possible to shift the balance of cell fate determination away from apoptosis and toward adaptive XBP1 signaling, with potential therapeutic implications.